Ca2+ regulates many biological processes including neuronal signaling, muscle contraction, and cell development and proliferation. Depending on their intracellular location, Ca2+ signals vary in amplitude and duration, together forming a complex Ca2+ signaling code. The endoplasmic reticulum (ER) functions as the primary intracellular Ca2+ store and is the site of protein synthesis and processing. Disruption of ER Ca2+ homeostasis triggers the ER stress response, a source of cell death signals. The release of Ca2+ from ER stores results in a rapid increase in [Ca2+]c and the released Ca2+ binds to a number of intracellular Ca2+ sensing proteins, such as calmodulin (CaM) and troponin C (TnC), as well as ion channels and enzymes to regulate a variety of cellular events and processes. Several human diseases, including various cardiomyopathies, Alzheimer's disease, cancer, and lens cataract formation are known to be associated with altered Ca2+ signaling and altered Ca2+ regulation by the ER store.
Because of the essential role of the ER in Ca2+ signaling, the determination of free [Ca2+]ER and its dynamic changes during cell signaling has attracted extensive interest. However, the lack of tractable biological Ca2+ indicators with affinities in the high μM to mM range has made it difficult to directly assess changes in [Ca2+]ER. Current estimates of [Ca2+]ER have been derived using three major types of Ca2+ indicators and sensors (18-23). These are: (1) synthetic small molecule fluorescent indicators such as Mag-Fura-2, (2) specifically modified derivatives of the chemiluminescent protein (photoprotein) aequorin, and (3) fluorescent indicators based on green fluorescence protein (GFP) variants with CaM or TnC. Although some small molecule dyes will accumulate in certain cellular compartments of cells, they cannot be unambiguously targeted to specific intracellular locations. Invasive methods are frequently required to eliminate the large fluorescence background resulting from the presence of these dyes in the cytosol. Targeted aequorins overcome some of the problems associated with the compartmentalized dye approach, but the low light output of these probes and the requirement for a soluble co-factor (coelenterazine) limit their usefulness.
GFP-based Ca2+ sensors, such as cameleons and pericams originally engineered by the groups of Miyawaki, Persechini, and Tsien, are based on either fluorescence resonance energy transfer (FRET) between two different GFP variants, or the effect of a Ca2+-dependent conformational change in natural Ca2+ sensing proteins (e.g. CaM) on the protonation state of the chromophore of a single GFP variant. Because these sensors are based on naturally-occurring essential Ca2+ binding proteins, a perturbation of the cellular environment through their introduction cannot be excluded, although many efforts have been made to decrease this possibility. Thus, there is a need to develop Ca2+ sensors that minimally compete for Ca2+ with existing cellular Ca2+ binding proteins and/or its target proteins, show robust Ca2+ responses, and exhibit Ca2+ binding affinities comparable to that of different cellular compartments, such as the ER. Accordingly, there is a need for improved analyte sensors and methods for measuring and detecting analyte activity.